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Research Article

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

View ORCID ProfileRyuta Kanai, View ORCID ProfileFlemming Cornelius, View ORCID ProfileHaruo Ogawa, Kanna Motoyama, View ORCID ProfileBente Vilsen, and View ORCID ProfileChikashi Toyoshima
  1. aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;
  2. bDepartment of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark

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PNAS January 5, 2021 118 (1) e2020438118; https://doi.org/10.1073/pnas.2020438118
Ryuta Kanai
aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;
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Flemming Cornelius
bDepartment of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark
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Haruo Ogawa
aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;
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Kanna Motoyama
aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;
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Bente Vilsen
bDepartment of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark
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Chikashi Toyoshima
aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;
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  • For correspondence: ct@iqb.u-tokyo.ac.jp
  1. Contributed by Chikashi Toyoshima, November 12, 2020 (sent for review October 1, 2020; reviewed by Kathleen J. Sweadner and Howard S. Young)

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Significance

Cardiotonic steroids (CTSs) are specific inhibitors of Na+,K+-ATPase and have been studied over a long period of time because of their potential therapeutic use in heart failure, hypertension, and cancers. Their affinities and binding kinetics are vastly different and seemingly rather inconsistent in the literature. Recent development of nonconventional CTSs with unexpected behaviors has revived interest in CTSs as potential drugs. In this report, we carried out a systematic study on various CTSs, including rostafuroxin and istaroxime, using kinetics and X-ray crystallography of their complexes with Na+,K+-ATPase, and explain how CTSs block the reaction cycle and why such large differences in inhibitory behaviors arise. The analysis suggests how isoform specificity may be achieved in order to improve their clinical usability.

Abstract

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3− complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

  • cardiotonic steroids
  • Na+,K+-ATPase
  • crystal structures
  • isoform specificity
  • sodium pump

Footnotes

  • ↵1To whom correspondence may be addressed. Email: ct{at}iqb.u-tokyo.ac.jp.
  • Author contributions: F.C., B.V., and C.T. designed research; R.K., F.C., H.O., K.M., B.V., and C.T. performed research; R.K., F.C., H.O., K.M., B.V., and C.T. analyzed data; and F.C. and C.T. wrote the paper.

  • Reviewers: K.J.S., Massachusetts General Hospital/Harvard Medical School; and H.S.Y., University of Alberta.

  • The authors declare no competing interest.

  • This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2020438118/-/DCSupplemental.

Data Deposition.

The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB ID codes 7D91–7D94 and 7DDF–7DDL).

Published under the PNAS license.

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Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state
Ryuta Kanai, Flemming Cornelius, Haruo Ogawa, Kanna Motoyama, Bente Vilsen, Chikashi Toyoshima
Proceedings of the National Academy of Sciences Jan 2021, 118 (1) e2020438118; DOI: 10.1073/pnas.2020438118

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Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state
Ryuta Kanai, Flemming Cornelius, Haruo Ogawa, Kanna Motoyama, Bente Vilsen, Chikashi Toyoshima
Proceedings of the National Academy of Sciences Jan 2021, 118 (1) e2020438118; DOI: 10.1073/pnas.2020438118
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